The present invention relates to enzymatic systems for carbon fixation and methods of generating same.
Photosynthesis is a process executed by photosynthetic organisms by which, inorganic carbon (Ci), such as CO2 and HCO3, is incorporated into organic compounds using the energy of photon radiation. Photosynthetic organisms, such as, soil-grown and aquatic plants and cyanobacteria (blue-green algae), depend on the organic compounds produced via photosynthesis for sustenance and growth.
In the process of transforming sunlight into biological matter, plants absorb ten times more carbon dioxide from the atmosphere than is emitted by the global human population. Moreover, agriculture, which is dependent on carbon fixation, consumes over 70% of the fresh water utilized by humanity and the majority of cultivatable land resources on earth. These figures point to the central place that carbon fixation by plants plays in our global ecological footprint. In nature the growth limiting factors of photosynthetic organisms vary between habitats and often include the availability of water, light, fixed nitrogen, iron and phosphorous. However, under human cultivation the usage of fertilizers and irrigation can make the carbon fixation rate limiting; for example, various C3 plants have shown a significant increase in growth rate when exposed to twice the atmospheric CO2 concentration.
Previous growth enhancements have been demonstrated by addressing several biochemical limiting factors, related both to the light-dependent and light-independent reactions. For example, transgenic Arabidopsis plants that expressed an efficient bacterial photorespiration pathway, instead of their natural photorespiration pathway, grew faster, produced more shoot and root biomass, and contained more soluble sugars [Kebeish R, et al. (2007) Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana. Nat Biotechnol 25(5):593-599]. In another effort, tobacco plants overexpressing sedoheptulose-1,7-bisphosphatase, an enzyme operating in the reductive pentose phosphate cycle (rPP, also known as the Calvin-Benson Cycle), were characterized by an increased photosynthetic rate and a 30% enhancement in biomass yield [Lefebvre S, et al. (2005) Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development. Plant Physiol 138(1):451-460].
The rPP cycle (FIG. 5A), used by the vast majority of autotrophic organisms for CO2 assimilation, is limited by the slow rate of Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase). The inverse correlation between the enzyme turnover number (˜2-4 s−1) and its CO2 specificity indicates that the enzyme might already be naturally optimized. Therefore, further optimization of Rubisco may prove difficult and lead to only marginal results [Raines C A (2006) Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle. Plant Cell Environ 29(3):331-339] thereby limiting the potential for increasing the rate of the rPP cycle. Designing and developing alternative (Rubisco independent) pathways that can support carbon fixation with a higher rate can therefore be highly beneficial.
To date, five natural metabolic pathways have been identified that are capable of performing carbon fixation in place of the classic rPP cycle. These are the reductive tri-carboxylic-acid (rTCA) cycle, postulated in the 60's; the oxygen sensitive reductive acetyl-CoA (rAcCoA) pathway; the extensively researched 3-hydroxypropionate (3-HP) cycle; the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle and the recently discovered dicarboxylate/4-hydroxybutyrate (DC/4-HB) cycle.